Ultraconservation identifies a small fraction of elements that are under similar evolutionary constraint. (a) We considered nucleotide substitutions in 256 non-exonic human-rodent ultraconserved elements in five additional placental mammalian genomes (chimpanzee, rhesus, dog, horse and cow). We found that 203 elements (79%) have at least one position substituted in other mammals, 153 (60%) have two or more substituted positions and 43 (17%) have substitutions at five or more positions. We did not consider imperfect sequence conservation due to insertions and deletions. (b) We identified more than 2,600 extremely constrained human-rodent elements at a constraint score threshold of ≥40, of which more than 2,300 are not defined as ultraconserved. Of the 500 most human-rodent constrained noncoding elements (score ≥74.7), 350 (70%) do not contain or overlap regions of ultraconservation. The graph does not depict overlap with possibly exonic ultraconserved regions. (c) Enrichment near genes involved in transcriptional regulation, general development and nervous system development. We considered the function (Gene Ontology, biological process) of the closest neighboring gene of each conserved element, and compared the observed numbers of genes in each category to the number expected based on all annotated RefSeq genes. Additional significantly enriched categories are listed in .

Highly constrained enhancers target expression to similar tissues independent of ultraconservation. (a) Binning of patterns driven by ultraconserved (top) and highly constrained human-rodent (bottom) enhancers into broad anatomical domains does not show significant differences for any structure (all P values > 0.05, Fisher’s exact test with Bonferroni correction for multiple hypothesis testing). Enhancers targeting expression to more than one region are reported in each respective category. (b) Examples of extremely constrained enhancers that contain (left) or do not contain (right) regions of ultraconservation, but drive highly similar expression patterns. Arrows indicate viewing angle of insets. Only one representative embryo per enhancer is shown; all patterns were reproducible in at least two additional embryos resulting from independent transgenic integration events. DRG, dorsal root ganglia. Genomic coordinates for all enhancers are provided in Supplementary Tables and .